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A series of two‐component powders and tableted mixtures of corn starch and lactose‐monohydrate were prepared and studied using the fiber optic sensor of a LabRam dispersion Raman microscope in order to establish a convenient method for quantitative determination of their concentration based on Raman spectra. The intensities of certain characteristic peaks of the components were used for calculating the calibration curve. Linear calibration curves were found in the concentration range of 10–80% with correlation coefficients of 0.9979–0.9996 for powders and tablets. Chemical mapping of the tablets was also carried out, thus allowing determination of the distribution of the components. Copyright © 2003 John Wiley & Sons, Ltd.

A series of two-component powders and tableted mixtures of corn starch and lactose-monohydrate were prepared and studied using the fiber optic sensor of a LabRam dispersion Raman microscope in order to establish a convenient method for quantitative determination of their concentration based on Raman spectra. The intensities of certain characteristic peaks of the components were used for calculating the calibration curve. Linear calibration curves were found in the concentration range of 10–80% with correlation coefficients of 0.9979–0.9996 for powders and tablets. Chemical mapping of the tablets was also carried out, thus allowing determination of the distribution of the components. Copyright © 2003 John Wiley & Sons, Ltd.

Infrared and Raman spectra of pinosylvin were recorded and the vibrational frequencies with the corresponding infrared intensities were compared with the results of ab initio calculations utilizing the DFT method with the Becke3P86 functional and the 6-31G(d) basis set. Normal coordinate analysis was carried out. The effect of the conformation of the OH groups on the distribution of net charges, molecular energy and vibrational fundamentals were analyzed. One of the OH-cis−OH-trans conformers has the lowest energy. The conformation has a strong effect on the aforementioned properties, e.g., the cis-to-trans transition generates electron repulsion toward the vinylidene group between the two benzene rings. The changes in the different properties are in good accordance with each other. For comparison, the vibrational spectra were also recorded and calculated for the parent compound, trans-stilbene.

GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis, cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanusand Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilisand the higher plant Mucuna hassjousing primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique. A number of regions were found to be strictly conserved between unrelated eukaryotes. These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia colienzyme.

GTP cyclohydrolase I catalyzes the initial and rate limiting step of the biosynthesis of tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylation. The mouse mutant HPH-1, previously generated by chemical mutagenesis, shows a phenylketonuria due to decreased hepatic GTP cyclohydrolase I activity. We show that both parameters GTP cyclohydrolase I activity and tetrahydrobiopterin synthesis significantly increase after weaning, but remain reduced during the lifetime. In the wild type mouse (C57BL/6), interferon-γ and kit ligand induce GTP cyclohydrolase I activity in primed T-cells and in bone marrow-derived mast cells, respectively. The same is true for the HPH-1 mutant, but the absolute values remain lower throughout. The open reading frame of GTP cyclohydrolase I is not affected by the hph-1mutation as shown by sequencing. Northern blot analysis demonstrates a marked decrease in the steady state mRNA level specific for GTP cyclohydrolase I.

Three different extruders, the Alexanderwerk gravity-feed roll extruder, the Gabler axial, single-screw extruder, and the NICA radial-screw extruder, were compared for their suitability for different placebo formulations and for fenoldopam pellets. A fourth extruder, the experimental ram extruder, was also included in some of the comparisons. The successful spheronization of extrudates from each of these extruders requires the correct water content. This water content, however, is different for each of the formulations and for each extruder. Generally, the Gabler unit required the highest amounts of water for a successful spheronization, yielding ≥90% between 710 and 1250 um. The NICA unit needed much less water for the same formulation, and the Alexanderwerk unit required even less water than the NICA unit. Pellet sphericity was also strongly dependent on the correct water content of formulations, but was generally better for pellets produced with the Alexanderwerk or NICA units. A two-way ANOVA test for the individual formulations showed a significant difference in the mean particle size of batches produced with the NICA or the Alexanderwerk and the Gabler extruder. No significant differences could be found between any of the Alexanderwerk or NICA batches. Both extruders showed a linear dependence of the mean particle size on the water content of formulations, but the Gabler extruder showed an almost unchanged particle size over a wide range of water contents, provided that the formulation could be extruded successfully.Batches that were extruded on the NICA unit showed a significantly lower bulk density than comparable Alexanderwerk or Gabler batches. Comparing the true density of pellets, we found that significant differences could only be stated for Avicel PH 101 + water batches and only for the NICA/Gabler interaction. True density increased for all three extruders with increasing amounts of soluble components and with increasing water content. The NICA batches also exhibited a significant difference of the Hausner factor from the other two extruders, but no differences could be found in the friability of pellets.

Three different extruders (the Alexanderwerk gravity feed roll extruder, the Gabler axial, single-screw extruder, and the NICA radial-screw extruder) were compared for their suitability for different placebo formulations and for fenoldopam pellets. A fourth extruder, the experimental ram extruder, was also included in some of the comparisons. Evaluation of the extrusion behavior of the three extruders showed differences as well as similarities among them, depending on the composition of the formulation. Although the NICA and Alexanderwerk units extruded all formulations successfully, the Gabler extruder failed to do so at a content of 60% of soluble ingredients, such as lactose or mannitol. The extrudate surface improved for all extruders with an increase in water content of formulations, but was generally smoother for the Gabler than for the NICA or the Alexanderwerk units. A formulation with colloidal Avicel as spheronization aid showed an identical extrusion behavior for all of the investigated extruders. Of the three extruders, the Gabler unit showed the highest heat generation during extrusion, especially when extruding formulations with a low water content or high contents of soluble ex-cipients. However, when the loss of water during extrusion or spheronization for various formulations was compared, only a two-way ANOVA test on the differences between the water content after extrusion and after spheronization showed a statistically significant difference between the Alexanderwerk or NICA and the Gabler extruder. The two-way ANOVA also proved that this difference is significant only for some formulations, e.g., lactose + Avicel PH 101 formulations, but not for Avicel PH 101 formulations.

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

Bone marrow aplasia was induced by fractionated whole body irradiation with 3000 R and restitution was started by autologous bone marrow transplantation. During the period of aplasia the amount of buffy coat biopterin clearly followed the decline of leukocytes and, vice versa, during reconstitution it largely paralleled their increase in number. The amount of red cell biopterin closely correlated with the number of reticulocytes rather than with the fairly constant values for hematocrit or of erythrocytic protein. Thereby it clearly followed the various periods of red cell recovery. The amount of cellular biopterin, its concentration with respect to cell number or to unit of protein and the percentage distribution of biopterin among the cell fractions are presented as characteristics of the activity of hemopoietic cell proliferation.

Using the Crithidia assay 3.0 ng biopterin/ml blood was found, of which one third was present in the plasma. The erythrocyte fraction comprised 1.7 ng and the buffy coat 0.33 ng. After Ficoll separation 0.050 ng were found in the lymphocyte layer of 1 ml blood. During blast crisis of chronic myelocytic leukemias increased amounts of biopterin were found in the erythrocyte fraction and in the buffy coat. The high biopterin concentration per unit of protein in the white cell fraction indicated the presence of blasts. In Polycythemia vera increased amounts of biopterin in both the red cell fraction and in the buffy coat were also found but the percentage distribution within total cellular biopterin was markedly shifted toward the erythrocyte fraction. In cases of chronic and acute lymphocytic leukemias the low amounts of biopterin in the red cell fraction agreed with the current view of partial extinction of the erythropoietic line. The isolated lymphoblasts were characterized by high biopterin concentrations per unit of protein. During remission the biopterin patterns approached normal levels.

The control of (6R)-5,6,7,8-tetrahydrobiopterin (H4biopterin) synthesis in primed T cells was analyzed by using the human T cell leukemia virus type I (HTLV-I)-transformed T cell line MT-2. In contrast to the slowly progressing induction of H4biopterin synthesis during activation of resting T cells, it is completed during a 59-h period and is directed by a synergism of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Both GTP cyclohydrolase and (6R)-(1‘,2‘-dioxopropyl)-5,6,7,8-tetrahydropterin synthase activities are induced by IFN-gamma. They are further enhanced by combined treatment with IL-2, which per se is ineffective. Furthermore, the combined treatment synchronizes the time periods of both maximum activities, now extending from 33 to 44 h. This period correlates with high cellular H4biopterin levels. It is preceded by a fast and transient period of H4biopterin increase which depends on the synergistic action of both IFN-gamma and IL-2. It coincides with a transient increase in sepiapterin reductase activity. In contrast to MT-2 cells, HTLV-I-transformed HUT 102 cells constitutively secrete IFN-gamma and express IFN-gamma mRNA. The accumulation of H4biopterin is suppressed by anti-IFN-gamma polyclonal antibody and correlates with constitutive expression of all H4 biopterin-synthesizing enzymes.

GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hültner, L. , Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, W. (1993) J. Biol. Chem. 268, 12544-12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivo labeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.

1.The increase in the activity of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase by irradiance and its reversal by darkness were measured. AT-values (activity after irradiance: activity in the dark after complete loss of activation) were plotted against increasing time of irradiation as well as against increasing time of darkening.2.The AT-values for irradiance as well as for darkening yield sigmoidal types of curves, and are similar to those corresponding to the integral of a curve of the type y=e-x2(Gauss-curve). The (theoretical) sigmoidal curve was transformed (by double variable transformation) to optimally cover the experimental data. By differentiation Gauss-curves are obtained which express the change of enzyme activity per unit of time. The maximal points of these curves correspond to the half maximum of activation (not activity!) and to the highest change of activity per unit of time.3.Figs. 3, 4 and 5 show that the increase in activity after onset of irradiation starts earlier than does the decrease after onset of darkening. Conditions which may cause the preservation of the activated enzyme during about 100 sec after darkening are discussed. However, the decrease is much more rapid later on than is the increase in the light; the change in activity (change of AT) per unit of time is smaller in the activation process than it is in the inactivation process. Thus the Gauss-curve becomes much sharper in the latter case.4.Constant irradiance (irradiation with constant Eeduring a given time period) at different points of dark-inactivation causes an activation velocity which increases with the time of the preceding dark period (Fig. 6). Therefore the Gauss-curve becomes sharper with increasing time of the preceding dark period (Fig. 7).5.Increasing irradiance (irradiation with constant Eeduring an increasing time period) at 3 different points of inactivation by variation of the time of predarkening also results in an increase in activation velocity with increase of time of predarkening (Figs. 8 and 9).6.The model which best fits earlier experimental results as well as the kinetic studies is one in which activation is accomplished by association of four subunits of the enzyme (Eo) to the activated tetrameric form (Eakt). NADPH, created during the non-cyclic electron flow, could be the allosteric effector. Thus the activation velocity (change in activity per unit of time) is dependent on the concentration of Eo. Both increase with increasing time of darkness. In contrast the time period of activation at constant irradiation is independent of the concentration of Eo and is only dependent on the amount of the effector (NADPH), which increases with increasing time (increasing irradiance).

1.In the green alga Acetabularia mediterranea illumination increases the activity of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase compared with the dark value just as it does in the leaves of higher plants.2.One day after enucleation of the Acetabularia-cell (by removal of the rhizoids) no enzyme activity is detectable either in dark or in light. Five days after the treatment the dark value of the enzyme activity reaches approximately the level of the intact plants, and illumination again increases the activity. It is concluded that the effect is independent of the presence of the nucleus.

1.NADP+-dependent GPD was clearly active in 8 out of 13 Rhodophyta and in 5 out of 8 Phaeophyta tested.2.NAD+-specific GPD was detectable in all of the 6 red algae analysed and in the 3 brown algae tested.3.Aldolase-activity was found unequivocally in 8 out of 10 red algae and in 7 out of 8 brown algae.4.It is concluded that the absence of these enzymes is not a characteristic for these groups of algae.5.Light increases the activity of the NADP+-dependent GPD in species of Chlorophyta and Rhodophyta, and probably also in species of Phaeophyta.

In elf verschiedenen Gattungen aus sechs Klassen der Algen wurde neben den Streptococcus faecalis-aktiven Verbindungen der Folsäure-Gruppe im Wachstumstest mit Crithidia fasciculata “unkonjugiertes” Pterin nachgewiesen. Dieses besitzt — als Voraussetzung für seine Wirksamkeit gegenüber Crithidia — an Stelle der p-Aminobenzoylglutaminsäure eine Polyhydroxyseitenkette mit mindestens 2 OH-Gruppen in cis-Stellung. An teilweise gereinigten Extrakten sowie an einem weitgehend gereinigten Präparat aus Spirogyra wurde seine Abgrenzung gegenüber den Folsäure-Verbindungen gesichert.